Research Article www.acsami.org
Facile Assembly of Functional Upconversion Nanoparticles for Targeted Cancer Imaging and Photodynamic Therapy Liuen Liang,†,‡ Andrew Care,‡,§ Run Zhang,‡,§ Yiqing Lu,‡ Nicolle H. Packer,‡,§ Anwar Sunna,‡,§ Yi Qian,† and Andrei V. Zvyagin*,‡,⊥ †
Department of Biomedical Sciences, §Department of Chemistry and Biomolecular Sciences, and ‡ARC Centre of Excellence for Nanoscale BioPhotonics, Macquarie University, North Ryde, New South Wales 2109, Australia ⊥ Laboratory of Optical Theranostics, N. I. Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod 603950, Russia S Supporting Information *
ABSTRACT: The treatment depth of existing photodynamic therapy (PDT) is limited because of the absorption of visible excitation light in biological tissue. It can be augmented by means of upconversion nanoparticles (UCNPs) transforming deep-penetrating near-infrared (NIR) light to visible light, exciting PDT drugs. We report here a facile strategy to assemble such PDT nanocomposites functionalized for cancer targeting, based on coating of the UCNPs with a silica layer encapsulating the Rose Bengal photosensitizer and bioconjugation to antibodies through a bifunctional fusion protein consisting of a solid-binding peptide linker genetically fused to Streptococcus Protein G′. The fusion protein (Linker-Protein G) mediates the functionalization of silica-coated UCNPs with cancer cell antibodies, allowing for specific target recognition and delivery. The resulting nanocomposites were shown to target cancer cells specifically, generate intracellular reactive oxygen species under 980 nm excitation, and induce NIR-triggered phototoxicity to suppress cancer cell growth in vitro. KEYWORDS: photodynamic therapy, upconversion nanoparticles, targeted imaging, luminescence resonance energy transfer, solid-binding peptides
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INTRODUCTION Existing cancer treatments, including chemotherapy and radiotherapy, suffer from concomitant side effects. Clinically accepted light-based photodynamic therapy (PDT) offers a unique localized treatment with mild side effects and can supplement the other regimens. PDT stands out because of its reduced invasiveness, high sensitivity, site specificity,1,2 and immunogenicity capable of driving antitumor immunity,3 in contrast with the immunosuppressive effects of chemotherapy and radiotherapy. PDT is based on the photochemical reactions of photosensitizers (PSs) to generate cytotoxic reactive oxygen species (ROS) toward cancer cells.4 The majority of PSs currently used in PDT are excited by light in the ultraviolet (UV) or visible range, where the treatment penetration depth in biological tissues ranges from 60 μm (at 340 nm) to several millimeters at the far-red wavelength band.5 This limitation largely precludes PDT treatment of tumors with sizes of more than 1 cm3.6 In contrast to UV and visible light, light of the near-IR (NIR) wavelength from 650 to 1350 nm has a larger penetration depth in biological tissues,7,8 providing a promising approach to improving the treatment depth for PDT.9 In particular, increasing attention has been paid to the lanthanide-doped upconversion nanoparticles (UCNPs) that are capable of © 2016 American Chemical Society
converting NIR excitation to visible/UV emission, as light transducers to activate clinically approved PSs.10−12 Depending on PS absorption, UCNPs can be engineered to have optimized emission overlapping with the absorption band of the PS, which facilitates the process of energy transfer and makes such NIRmediated PDT highly efficient.13−16 Additionally, UCNPs offer exceptional photostability and background-free imaging capability, which is ideal for high-contrast biomedical imaging.17−21 Therefore, a hybrid nanocomposite combining UCNPs and PSs holds great promise for image-guided diagnosis and PDT. To enable targeted PDT treatment with minimum side effects as well as direct visualization of the tumor, UCNP nanocomposites are required to bioconjugate to active tumortargeting moieties such as folic acid,22−24 peptides,25 and antibodies.26 Unfortunately, the conventional bioconjugation techniques (e.g., amine-reactive cross-linking) used in these reports often lack sufficient control over the functional display of the immobilized targeting moiety, which may prevent selective molecular recognition between the targeting moiety and its receptor/molecular partner. In addition, these biohybrid nanoReceived: January 19, 2016 Accepted: April 27, 2016 Published: April 27, 2016 11945
DOI: 10.1021/acsami.6b00713 ACS Appl. Mater. Interfaces 2016, 8, 11945−11953
Research Article
ACS Applied Materials & Interfaces
Scheme 1. Schematic Illustration of (a) the Fabrication of UCNP@SiO2(RB) and Its Functionality and (b) LPG-Mediated Bioconjugation of UCNP@SiO2(RB) with Antibodies and Their Application in Targeted PDT
diamidino-2-phenylindole dihydrochloride (DAPI), and paraformaldehyde (PFA) were obtained from Sigma-Aldrich Chemicals (Sydney, Australia). Rose Bengal (RB) was purchased from Alfa Aesar (China). Purified mouse antihuman CD326 (EpCAM; clone HEA-125) was purchased from Miltenyi Biotech (Sydney, Australia). Recombinant LPG was produced in Escherichia coli and purified by ion-exchange chromatography as described previously.27 Characterization Instruments. The absorbance spectra of RB were obtained using a UV/vis spectrophotometer (Cary 5000, Varian). Transmission electron microscopy (TEM) measurements were performed with a Philips CM10 transmission electron microscope operating at 100 kV. The size distribution of UCNPs was analyzed using ImageJ. The high-resolution TEM (HRTEM) image was recorded using a JEM-3000F transmission electron microscope operating at 300 kV. Emission and excitation spectra were acquired using a Fluorolog-Tau3 spectrofluorometer (Jobin Yvon-Horiba) equipped with an external 978 nm continuous-wave diode laser with maximum achievable power of ∼1.2 W. The ζ-potential and dynamic light scattering measurements were carried out using a Zetasizer Nano ZS90 (Malvern Instruments Ltd.). Synthesis of Core−Shell (NaYF4:Yb,Er@NaGdF4) Nanoparticles. Core β-NaYF4:Yb,Er nanoparticles were synthesized following a protocol developed previously.28 Typically, for the synthesis of NaYF4:Yb(18%),Er(2%) nanoparticles, 0.8 mmol of YCl3 was mixed with 0.18 mmol of YbCl3 and 0.02 mmol of ErCl3 with 6 mL of OA and 15 mL of ODE in a 100 mL three-neck round-bottomed reaction flask. The mixture was heated to 160 °C for 30 min under an argon flow. The resulting mixture formed a light-yellow solution and was cooled to room temperature (RT). Afterward, a methanol solution containing NH4F (0.148 g) and NaOH (0.1 g) was added, and the resulting solution was stirred for 30 min. The solution then was heated slowly to 110 °C under an argon flow for 30 min to completely remove methanol along with some water. After this step, the reaction mixture was heated to 310 °C for 1 h with constant stirring. Finally, nanoparticles were precipitated with ethanol and washed several times with ethanol/methanol (1:1, v/ v), and the resulting core UCNPs were redispersed in cyclohexane. The synthesis of NaYF4:Yb,Er@NaGdF4 nanoparticles was based on the Ostwald ripening-mediated method with some modification.29 First, cubic NaGdF4 nanocrystals were prepared as follows: 1 mmol of YCl3, 6 mL of OA, and 10 mL of ODE were mixed in a 50 mL reaction flask, and the resulting mixture was heated to 150 °C under an argon flow with constant stirring for 30 min to form a light-yellow solution that was
complexes are assembled via a chemical reaction between targeting molecules and nanoparticles, where an antibody can bind to more than one nanoparticle, leading to particle aggregation. Therefore, new strategies of assembling UCNPs with PSs and targeting moieties while maintaining their functionality and selectivity are highly desirable. In this work, we explore a facile strategy to assemble targeting UCNP−PS nanocomposites. As illustrated in Scheme 1, first, the green-emitting Yb3+/Er3+-codoped UCNPs are coated with a silica layer that encapsulates a common PS, Rose Bengal (RB), for PDT. Upon 980 nm irradiation, energy is transferred from the UCNPs to the neighboring RB molecules to activate the generation of singlet oxygen. Then, a bifunctional fusion protein, consisting of a silica-specific solid-binding peptide (referred to as the Linker, L) genetically fused to the N-terminus of Streptococcus Protein G′ (PG, an antibody-binding protein), is applied to facilitate oriented and sterically accessible immobilization of tumor-targeting antibodies onto the surfaces of the silicacoated nanocomposites. The Linker domain of Linker−Protein G (LPG) exhibits high binding affinity toward the silica surface, and the IgG-binding protein (PG) binds to the Fc fragment of IgG antibodies, thereby ensuring the functional display of conjugated antibodies and avoiding concurrent reactions between one antibody and different nanoparticles in crosslinking methods. Finally, these functionalized nanocomposites are validated for targeted imaging and selective killing of human colorectal adenocarcinoma HT-29 cells in vitro.
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EXPERIMENTAL SECTION
Reagents and Chemicals. All reagents were of analytical grade and were used as received without further purification. Yttrium(III) chloride hexahydrate (YCl3·6H2O; 99.999%), ytterbium(III) chloride hexahydrate (YbCl3·6H2O; 99.9%), erbium(III) chloride hexahydrate (ErCl3· 6H2O; 99.9%), sodium hydroxide (NaOH; ≥97.0%), ammonium fluoride (NH4F; ≥98.0%), oleic acid (OA; 90%), oleylamine (OM; ≥98.0%), 1-octadecene (ODE; 90%), cyclohexane (99.5%), tetraethyl orthosilicate (TEOS), Igepal CO-520, ammonium hydroxide solution (NH4OH; 30%), 1,3-diphenylisobenzofuran (DPBF), thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 4,611946
DOI: 10.1021/acsami.6b00713 ACS Appl. Mater. Interfaces 2016, 8, 11945−11953
Research Article
ACS Applied Materials & Interfaces
Figure 1. TEM images of (a) core UCNPs (NaYF4:Yb,Er; the inset is the HRTEM image of a single core nanoparticle) and (b) core−shell UCNPs (NaYF4:Yb,Er@NaGdF4). (c) Core−shell UCNPs with UCNP@SiO2(RB). The insets are photographs of UCNP@SiO2(RB) pelleted (left) and dispersed (right) in water. (d−f) Size distributions of the corresponding nanoparticles. (g) Upconversion luminescence spectra of core and core−shell UCNPs under 980 nm excitation. (h) Normalized excitation and emission spectra of UCNP@SiO2(RB) and free RB. cooled to RT. Second, 10 mL of a methanol solution containing 1.6 mmol of NH4F and 1 mmol of NaOH was added, and the solution wa stirred for 30 min. After heating to remove methanol, the solution was cooled, and 3 mL of OM was added. The solution was heated to 290 °C under an argon flow with vigorous stirring for 45 min and then cooled to RT. The NaGdF4 particles obtained were collected and resuspended in a mixture of 5 mL of OA, 8 mL of ODE, and 1 mL of OM and used as sacrificial nanoparticles. A total of 0.2 mmol of core UCNPs in 15 mL of cyclohexane was added to a 100 mL reaction flask and mixed with 10 mL of OA, 16 mL of ODE, and 2 mL of OM. After heating slowly to 110 °C for 30 min to remove cyclohexane, the solution was heated to 305 °C, and 300, 200, and 100 μL sacrificial nanoparticles were injected stepwise into the reaction with 10 min reaction ripening after each injection. The resulting core−shell NaYF4:Yb,Er@NaGdF4 nanoparticles were collected and washed with centrifugation and dispersed in cyclohexane for further silica coating. Synthesis of RB-Loaded Silica-Coated UCNP [UCNP@ SiO2(RB)]. A modified water-in-oil microemulsion method30 was used to prepare UCNP@SiO2(RB). In a typical procedure, 0.5 mL of Igepal CO-520 was dispersed in 5 mL of cyclohexane, followed by injection of a 0.1 mmol of core−shell UCNPs in 5 mL of cyclohexane solution into the mixture. After stirring for 3 h, 1 mg of RB was added to the mixture, and stirring was continued for another 2 h. A total of 500 μL of ammonia (30%) was then added, followed by slowly injection of 35 μL of TEOS (2 μL/min). The mixture was sealed and kept stirring for 24 h. The product was then precipitated and washed three times with ethanol, followed by washing with water three times and finally storage in water. LPG-Mediated Functionalization of UCNPs. UCNP@SiO2 (with and without RB) was functionalized with antibodies via LPG as follows. Nanoparticles (1 mg) were sonicated before use and then rinsed three
times with a 100 mM Tris−HCl buffer, pH 7.5. The washed particles were resuspended by sonication in the same buffer containing 30 μg of purified LPG and incubated with rotation at RT for 15 min. Particles were collected by centrifugation, and the unbound LPG was removed, after which the particles were washed two times. The LPG-coated particles were then incubated with 20 μg of antibody (referred to as Ab) with rotation at RT for 15 min. Particles were collected by centrifugation, and the unbound Ab was removed, followed by two additional washing steps. The resulting Ab-functionalized UCNPs finally were dispersed in 200 μL of phosphate-buffered saline (PBS) for use in subsequent experiments. Cell Imaging. Human colon adenocarcinoma HT-29 cells and murine microglial BV2 cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin under 5% CO2 at 37 °C in a humidified incubator. For the EpCAM-targeted imaging, 2.5 × 104 cells/well of HT-29 (high EpCAM expression) and BV2 (low EpCAM expression) cells were seeded into 24-well plates with a coverslip placed at the bottom of each well. After incubation for 24 h, both of the plates of cells were washed three times with PBS and fixed with a 4% PFA solution for 15 min at RT before washing again three times with PBS. The cells were incubated with a 25 μg/mL UCNP@SiO 2 (RB)-LPG-Ab PBS suspension at RT for 1 h, followed by DAPI nuclei staining for 10 min. Afterward, PBS was used to rinse the cells five times to wash away the unlabeled nanoparticles and DAPI. Finally, the coverslips were then mounted on glass slides and sealed with nail polish. Upconversion fluorescence imaging was performed with our in-house-built epiluminescence microscope equipped with a fiber-coupled 978 nm diode laser and a 405 nm laser to illuminate the UCNPs and DAPI. The filters used 11947
DOI: 10.1021/acsami.6b00713 ACS Appl. Mater. Interfaces 2016, 8, 11945−11953
Research Article
ACS Applied Materials & Interfaces in the microscopes include a high-pass absorbance filter (850 nm) placed in the excitation beam path, a dichroic beam splitter (511 nm), and an additional short-pass filter (842 nm) in the detection beam path. The overall emission of the nanocomposite in the range of 511−842 nm was detected by the camera and represents photoluminescence from UCNPs. Detection of Singlet Oxygen in Solution. Singlet oxygen generation was evaluated in a solution of UCNP@SiO2(RB)-LPG-Ab using DPBF as a 1O2 detection probe. Typically, 10 μL of a 10 mM DPBF ethanol solution was mixed with 1 mL of a 1 mg/mL UCNP@ SiO2(RB)-LPG-Ab aqueous suspension. The solution then was placed in a cuvette and irradiated with a 980 nm NIR laser at 1.5 W/cm2 for 30 min. The absorption of DBPF was collected every 5 min using a NanoDrop 2000c UV−vis spectrophotometer. For comparison, the same amounts of DPBF mixed with UCNP@SiO2(RB)-LPG-Ab in the dark and UCNP@SiO2-LPG-Ab with NIR treatment were measured as controls. Intracellular ROS Generation Detection. Intracellular singlet oxygen generation was detected with an Image-iT LIVE Green Reactive Oxygen Species Kit (Molecular Probes). HT-29 cells were seeded into coverglass−bottom confocal dishes at a density of 3 × 105 cells/dish and left to grow for 1 day at 37 °C in an incubator. Then the HT-29 cells were loaded with 100 μg/mL UCNP@SiO2(RB)-LPG-Ab and UCNP@SiO2-LPG-Ab for 12 h. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA; 25 μM) was loaded into the cells, which were incubated in darkness for 30 min. The cells then were washed twice with 1×HBSS and subjected to NIR irradiation for 5 min (with intermittent 1 min breaks after each 1 min of irradiation to prevent overheating) at a power density of 1.5 W/cm2. The cells were washed twice and incubated with 1×HBSS buffer. The production of singlet oxygen was visualized by an Olympus Fluoview FV1200 confocal microscope. Oxidized 2′,7′dichlorofluorescein (DCF) was excited with a 473 nm laser, and the emission was collected through a 490−590 nm filter. The intracellular ROS level was analyzed via the fluorescent intensity from DCF. HT-29 cells were seeded into 96-well plates until adherent and then incubated with 100 μg/mL UCNP@SiO2(RB)-LPG-Ab and UCNP@SiO2-LPG-Ab for 12 h. After treatment similar to that mentioned above, the fluorescent intensity of the cells in different treatments was recorded with excitation at 485 nm and emission at 520 nm using a PHERAstar microplate reader. In Vitro Cytotoxicity Study. HT-29 cells were seeded in a 96-well culture plate at a density of 5000 cells/well for in vitro cytotoxicity assays. After being incubated for 12 h, the cells were loaded with UCNP@SiO 2, UCNP@SiO 2(RB), UCNP@SiO2(RB)-LPG, and UCNP@SiO2(RB)-LPG-Ab at serial concentrations of 0, 12.5, 25, 50, 100, and 200 μg/mL with five parallel wells for each concentration. Treated cells were kept incubated in the dark for another 24 h. The standard MTT assay was carried out to determine the cell viabilities relative to untreated cells. Photodynamic Effect of UCNP@SiO2(RB)-LPG-Ab on Cancer Cells. HT-29 cells were incubated with different concentrations of UCNP@SiO2(RB)-LPG-Ab (0, 100, and 200 μg/mL) in a 96-well plate for 24 h at 37 °C in the dark to assay the EpCAM-targeted PDT effect. After removal of the medium containing noninternalized nanoparticles, the cells were washed three times with PBS and then irradiated with NIR light (1.5 W/cm2; 1 min interval breaks to avoid overheating) in fresh culture media for 0, 5, 10, and 15 min. After NIR treatment, the cells were incubated for a further 12 h in the dark before viability evaluation with MTT assay.
phase NaYF4. To suppress surface-related quenching of the upconversion emission, an inert shell of NaGdF4 (∼1 nm thickness) was further deposited onto the core UCNPs. The core−shell UCNPs (NaYF4:Yb,Er@NaGdF4) retained their morphology and dispersion (Figure 1b) and had an average diameter of 31 ± 1 nm (Figure 1e). Compared to the core UCNPs, the core−shell UCNPs exhibited 1.7-fold enhanced upconversion photoluminescence under continuous-wave 980 nm excitation (Figure 1g). Silica coating is commonly applied to UCNPs to improve their aqueous solubility and stability in physiological environments. It can also serve as reservoirs for storing substantial quantities of therapeutic drugs, including PSs.31−34 A thin layer of silica (SiO2) was coated onto the core−shell UCNPs using a water-in-oil microemulsion method. The RB molecules were loaded into the silica layer simultaneously during the coating process. The resulting UCNP@SiO2(RB) particles were spherical in shape, with a mean diameter of 43 ± 2 nm (Figure 1c,f). The core−shell UCNPs were individually enclosed by a uniform layer of silica, with a thickness of approximately 6 nm as measured by TEM. This was within the critical distance required for efficient luminescence resonance energy transfer (
